The Ti–22Al–25Nb alloy, which possesses a complex microstructure, is a new type of lightweight high-temperature structural material. Revealing the effects of microstructure and temperature on the mechanical properties of this alloy is crucial for applications in the aerospace industry. In this study, we designed three types of microstructures of the Ti–22Al–25Nb alloy, namely, single-phase B2, dual-phase α2 + B2, and triple-phase O + α2 + B2, and investigated their deformation and fracture behavior with increasing temperature (25,750,and 930 °C). The O + α2 + B2 sample possessed the best comprehensive mechanical properties, and the softening of the O phase at elevated temperatures was mainly due to massive {001}<110>O basal slip and twinning. The evolution of O phase twins with increasing temperature can be described as follows: (021) → (021) + (110) → (110). At 750 °C, (021) twins formed in the strip-shaped O phase in B2 grains, while (110) twins formed in the spheroidized O phase at B2 grain boundaries. In addition, B2 grain boundary embrittlement was observed at elevated temperatures, and its mechanism can be summarized as follows. Elevated temperatures induced element segregation at the grain boundaries between hypersaturated-state B2 grains, enriching Ti and Al and diffusing Nb into the B2 grains on the nanometer scale, leading to a thin-shell O phase with a thickness of tens of nanometers precipitating at the B2 grain boundaries. During elevated-temperature fracturing, cracks propagated quickly along the interface between the thin-shell O phase and B2 phase, leading to embrittlement of the B2 grain boundary. The precipitation of many fine granular O or α2 phases at the B2 grain boundaries can inhibit the precipitation of the thin-shell O phase and hinder the propagation of cracks along the grain boundaries. Therefore, the microstructure of this alloy should be controlled to precipitate many fine granular O or α2 phases to occupy the B2 grain boundaries and inhibit its elevated-temperature embrittlement.